[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .

Objective To improve detection rate and diagnostic accuracy of single ventricle(SV) in obstetric fetal echocardiography,to investigate the common types and complicated malformations of SV in the fetus,and to summarize the differential announcements in diagnosing fetal SV.Methods In 345 fetal hearts which were diagnosed as congenital heart disease by fetal echocardiography in our hospital,73 cases diagnosed as SV,including 3 cases appeared as ones of twins,were included in this study.Systemic scanning and multiple-views fetal echocardiography were used to examine these enrolled fetuses.Results In all 73 SV eases,3 cases were diagnosed as simple SV,the others were diagnosed as SV accompanied with other abnormalities,among them 44 cases accompanied with single atrium,18 cases with single atrium and persistent truncus arteriosus,2 cases with pulmonary atresia,20 cases with pulmonary artery stenosis,4 cases with partial atrioventricular septal defect,3 eases with aorta dysplasia or aortic valve dysplasia.SV types were classified as 24.7％ in type A,13.7％ in type B,46.6％ in type C and 15.0％ in type D respectively.68.2％ of the cases were diagnosed with aortic D-transposition,and 45.2 ％ with common inlet,42.5 ％ with single inlet and 12.3 ％ with double inlet respectively.42 SV cases were executed termination of pregnancy which 11 cases were confirmed by pathology and the other 31 cases were out of following-up.Conclusions Most cases of fetal SV were accompanied other abnormalities and simple SV was rare.Type C in which ventricular structure was combined with left and right ventricle was the most common type.To avoid the false diagnosis,much attention must be paid to distinguish big papillary muscle and abnormal muscle bundle from interventricular septum during ultrasonic examination.

Objective To investigatethe role of NF-κB played in the process of the cord blood monocytes differentiating into dendritic cells(DCs)induced by astragalus polysaccharide(APS)and to explore the signal transduction pathway involved in this process.Methods Umbilica]cord blood was collected in aseptic conditions.The cord blood monocytes were obtained by density gradient centrifugation and were divided into three groups afterwards.In the control group.cells were cultivated in the RPMI 1640 complete medium.In the APS group.cells were cultivated in the RPMI 1640 complete medium containing 100 mg/L APS.In the PDTC group:cells were treated with 10 μmol/L disulfide carbamate(PDTC).NF-κB inhibitor in 30 min followed by cultivalion in the RPMI 1640 complete medium containing 100 mg/L APS.,The morphological changes were observed during the process of cultivation by the optical microscope and transmission electron microscopy.Cells were collected 12 d later and the cellular immunophenotyping was assayed by FCM.,The activation and migration of NF-κB fluorescence in the cells was examined by the immunoflouresce microscopy.Results (1)Cells in the control group grown up without cluster forformation and were found fusiform and macrophage-like in 12 d.Cells in the APS group grown up in clnstem,and morphological changes were found from the circular shape to a typical dendritic cells-like shape.Cells in the inhibitor group grown up slowly and without cluster formation,and cell morphdogy had no significant change.(2)The expression of DCs-specific antigen CD80,CD83 and CD86 in the APS group was higher than that in the control group and inhibitom group(P<0.01).The expression of those antigen in the control group and PDTC group was similar and had no statistically significance(P>0.05).(3)NF-κB fluorescence in the nuclei was examined by the immunoflourescence microscopy and was much higher in the APS group than that in khe other groups,especially in 72 h with the activation rate of NF-κB (75.20±7.37)%,while(13.20±3.46)% of PDTC group and(8.20 ±1.92)%,respectively(P<0.01).Conclusion Astragalus polysaccharide can induce the differentiation of umbilical cord blood cells into DCs,and NF-κB is the key component of the signal transduction pathway involved in this process.

Objective To investigate the common types of fetal cardiac malformations and complicated malformations,and to assess the value of classifying on these types.Methods 3201 pregnant women were undergone with fetal echocardiography (FECG),239 fetuses of them were diagnosed to be suffered with congenital heart disease(CHD),and 8 cases were one of twins with abnormal heart confirmed by FECG.All new-births were examined by echocardiography within half year after their births.Results 155 complex CHD in 239 fetuses were diagnosed by FECG,in them the common malformations were in turn 59 cases with diagnosed univentricular heart,29 cases with double outlet right ventricle,19 with atrio ventricular septum defect,12 with tetralogy of Fallot or quinalogy of Fallot,11 with persistent truncus arteriosus,6 with right ventricular dysplasia syndrome,6 cases transposition of the great vessels.100 cases were induced labor,41 of them were comfirmed by pathology.16 fetuses were born,123 cases were being pregnanted or un-followed up.Conclusions Complex and multi-malformation were common in fetal cardiocascular abnormalities.Diagnosing rate of fetal CHD(FCHD) in our enroll fetuses was 7.47 ％,rate of complex CHD vs CHD was 64.85 ％.According different types of FCHD,able to be operated or not after birth,surgery methods,as well as prognosis evaluations,all FCHD cases were classified into three subtypes:curable type,curable palliative type and untreatable type.This newly viewpoint will help pregnant women and their family to make reasonable selection.

Objective:To investigate the expression of circular RNA circOMA1 in children with acute myeloid leukemia (AML) and to investigate the effect and mechanism of circOMA1 on the cell proliferation and apoptosis of human acute monocytic leukemia cells (THP-1).Methods:Bone marrow samples of 14 children with AML at the initial diagnosis and after complete remission were collected as the initial diagnosis group and remission group, and bone marrow samples from 10 children without tumor or malignant blood disease in the same hospital and the same period were enrolled as the control group.Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of circOMA1, miR-145 and myelocytomatosis viral oncogene homolog (MYC) mRNA in clinical AML samples and THP-1 cell line.The cells transfected with THP-1 were divided into groups, the cells transfected with circOMA1 alone (circOMA1 high expression group) were transfected with pcDNA3.1 empty vector cells as control (pcDNA3.1 control group); the cells co transfected with circOMA1 and miR-145 (circOMA1+ miR-145 group) were treated with pcDNA3.1 and miR-NC co transfected cells were used as control (pcDNA3.1+ miR-NC group, circOMA1+ miR-NC group). Cell counting kit (CCK8) was adopted to detect the effects of circOMA1 and miR-145 on the cell proliferation of THP-1.The effects of circOMA1 and miR-145 on cell apoptosis of THP-1 were detected using flow cytometry, and the effects of circOMA1 and miR-145 on MYC protein expression was detected via Western blot.The comparison between groups was analyzed by independent sample t-test or paired sample t-test, and the correlation was analyzed by Pearson correlation. Results:The expression of circOMA1 in the initial diagnosis group(4.408±3.607) was significantly increased compared with that in the control group (0.998±0.560) ( t=2.946, P<0.01); the expression of circOMA1 in remission group(1.582±0.950) was significantly decreased compared with that in the initial diagnosis group( t= 3.628, P<0.01). The THP-1 cell experiments showed that compared to the pcDNA3.1 control group, the expression of miR-145 in the circOMA1 high expression group decreased ( t= 4.21, P<0.05), cell proliferation was enhanced at 72 h and 96 h ( t=5.46, 7.40, all P<0.05), apoptosis was inhibited( t=6.44, P<0.01). The expression of MYC protein in circOMA1+ miR-NC group was higher than that of pcDNA3.1+ miR-NC group( t=5.72, P<0.01), the expression of MYC protein in circOMA1+ miR-145 group was lower than that in circOMA1+ miR-NC group ( t=4.56, P<0.05); at 72 h and 96 h, the cell proliferation level of circOMA1+ miR-NC group was higher than that of pcDNA3.1+ miR-NC group ( t=5.77, 7.30, all P<0.05), the level of cell proliferation in circOMA1+ miR-145 group was lower than that in circOMA1+ miR-NC group ( t=4.66, 6.17, all P<0.05); the apoptosis rate of circOMA1+ miR-145 group was higher than that of circOMA1+ miR-NC group ( t=4.25, P<0.05). circOMA1 expression was negatively correlated with miR-145 expression ( r=-0.62, P=0.016) and positively correlated with MYC gene expression ( r=0.64, P=0.013) in clinical samples. Conclusions:circOMA1 is highly expressed in children with AML, and can promote AML cell proliferation and inhibit apoptosis through miR-145/MYC pathway, which provides a basis for AML therapy and diagnosis.

Objective To study the effect of different breast milk enhancement strategies and the incidence of complications in premature infants.Method Premature infants whose gestational age less than 34 weeks and birth weight less than 2 000 g were prospectively enrolled from January 2017 to February 2018 at the Department of Neonatology of Huangshi Maternal and Child Health-Care Hospital.According to the odd even number at the end of the hospitalization admission number,participants were assigned into 50～＜70 ml/(kg· d) group and 70～＜90 ml/(kg· d) group,When the children reached the corresponding amount of breast-feeding to be given breast milk fortifier.The demographic information,incidence of complications,rate of weight gain,percentage of extrauterine growth retardation (EUGR) and decrease of Z score at discharge were compared between groups.Result A total of 140 cases were included,with gestational age (31.4±1.9) weeks and birth weight (1 402±213) grams.Among the participants,67 infants were assigned to 50～＜70 ml/(kg·d) group,and 73 infants were assigned to 70～＜90 ml/(kg·d) group.There was no statistical difference between two groups in gender,gestational age,birth weight,length,head circumference,rates of asphyxia,ratio of intrauterine growth retardation,Z score of weight at birth,age at which breast milk fortifiers were added,full enteral feeding time,duration of parenteral nutrition,average length of hospital stay and the time of restoration of birth weight (P＞0.05).The proportion of feeding intolerance in 50～ ＜70 ml/(kg· d) group was higher than that in 70～＜90 ml/(kg· d) group (11.9％ vs.4.1％),the difference was statistical significant (P=0.013).There was no statistical difference in other complications between the two groups (P＞ 0.05).The body weight increase rate of premature infants in 50～＜70 ml/ (kg· d) group was higher than that in 70～＜90 ml/(kg· d) group,and decrease of Z score at discharge in 50～＜70 ml/(kg· d) group was lower than that of 70～＜90 ml/(kg· d),the difference was significant (P＜0.05).Conclusion Adding breast milk fortifier earlier——when the breast feeding amount of 50～＜70 ml/(kg· d)——is more beneficial to the growth and development of premature infants,it also reduces the incidence of EUGR on discharge.However,during the feeding process,it was necessary to be aware of the complications.

Androgen deprivation therapy is one of the main treatments for prostate cancer patients. In recent years, many studies have revealed that androgen deprivation therapy increases the risk of cardiovascular disease, which leads to the cause of death alongside tumor-related deaths. The mechanism may be related to the elevation of testosterone levels, follicle-stimulating hormone levels and unstable atherosclerotic plaque. Standardized cardiovascular disease management in this population is a key issue to improve survival and prognosis. This paper summarizes a management plan covering 5 areas, including history collecting and data examination, assessment, referral, health education, and regimen selection.

Objective To screen stroke risks among middle-aged and elderly people in community outpatient clinics in Shanghai.Methods A stroke risk screening was conducted among people aged 45-75 years selected with convenient sampling method from 10 community health service centers in Putuo district,Yangpu district and Pudong New Area of Shanghai during January to July 2017.The questionnaire and clinical measurement were used for screening.Multivariate logistic regression analysis was used to analyze the risk factors in subjects with high stroke risk.Results In this study,1 094 individuals with high stroke risk were screened out from 1 750 participants (62.5％).The proportion of high risk cases was higher among men (66.7％,473/709) than that among women (59.7％,621/1 041),unmarried,divorced or widowed (75.0％,90/120)than married or cohabitants (61.6％,1 004/1 630),living alone (72.1％,70/97) than living with others (61.9％,1 024/1 653) (x5=8.969,8.571,4.081;P＜0.01).Compared with non-high-risk subjects,the high-risk subjects had higher BMI,systolic blood pressure,diastolic blood pressure,fasting blood glucose,glycated hemoglobin,total cholesterol,triglycerides,serum creatinine and homocysteine,and were more likely to have carotid plaques or stenosis,the difference was statistically significant (P＜0.05).Multiple logistic regression analysis showed that unmarried/divorce/widowed,high BMI,systolic blood pressure,fasting blood glucose,total cholesterol,and carotid plaque or stenosis were positively associated with high stroke risk(OR=2.015,1.173,1.013,1.456,1.139,1.026,2.103;P＜0.05).Conclusions The proportion of high stroke risk individuals among middle-aged and elderly people is higher in community general practice outpatient clinics in Shanghai.Patients with high BMI,systolic blood pressure,fasting blood glucose and total cholesterol,and carotid plaque or stenosis,as well as those unmarried/divorced/widower should be the subjects for stroke intervention.

Objective:To understand the distribution and epidemiological characteristics of chronic lymphocytic leukemia (CLL) in Hunan Province.Methods:According to the audit methods and evaluation criteria specified by the National Cancer Registration Center, the registration data of CLL reported by 24 tumor registries was included. Through the research method of retrospective analysis, the selected registry data was calculated and analyzed according to the year, administrative division, urban and rural areas, gender and age.Results:A total of 104 newly diagnosed CLL patients were diagnosed in Hunan Province from 2014 to 2015, with an average annual morbidity of 0.39/100, 000. The morbidity in 2014 and 2015 was 0.39/100, 000 and 0.39/100, 000, respectively. The annual average morbidity in Zhuzhou was 0.8/100, 000, which was the highest among municipalities. The annual average morbidity in Kaifu District of Changsha was 1.65/100, 000, which was the highest among district-level administrative divisions. The morbidity of urban was higher than that of rural (Urban vs Rural, P=0.006). The male to female morbidity was 1.7∶1. The cases were mainly concentrated in the 61-70-year-old population, accounting for 33.65% of all cases (35/104). There were 64 patients died of CLL in Hunan Province from 2014 to 2015, and the average annual mortality was 0.24/100, 000. The mortality in 2014 and 2015 was 0.22/100, 000 and 0.26/100, 000, respectively. The average annual mortality in Hengyang was 0.53/100, 000, which was the highest among municipalities. The average annual mortality in Furong District of Changsha was 0.74/100, 000, which was the highest among district-level administrative divisions. The mortality of urban was higher than that of rural but with no significant difference ( P=0.006). The male to female mortality rate was 1.4∶1. The deaths were mainly concentrated in the 71-80-year-old population, accounting for 29.69% of all deaths (19/64). Conclusions:The morbidity of CLL in Hunan Province is much lower than that of European and American populations, and it mainly occurs in the elderly people. It is more common in men. The morbidity of urban is higher than that of rural and morbidity in Zhuzhou is the highest. The death of CLL patients was mainly in middle-aged and elderly population, with more males. The mortality of urban is slightly higher than that of rural and the mortality in Hengyang is the highest.

Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.